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| Construction and Analysis of Yeast Two-hybrid cDNA Library of the SRBSDV-infected Midgut of White Backed Planthopper (Sogatella furcifera) |
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Abstract Southern rice black-streaked dwarf virus (SRBSDV), the genus Fijivirus in the family Reoviridae, is transmitted mainly by white backed planthopper (Sogatella furcifera, WBPH) in a persistent-propagative manner. The epithelial cells of the midgut is the initially infected and replicated tissue for SRBSDV during the whole infection route in its insect vector. The effective multiplication of the virus is the key factor to determine whether the virus can be transmitted. In this study, to investigate the interaction between SRBSDV and its insect vector, and the mechanism of midgut protein regulating viral proliferation to transmit virus successfully. The yeast two-hybrid cDNA library for the midgut of SRBSDV-infected WBPH was constructed through SMART technique. The midgut of WBPH was dissected firstly. The mRNA was isolated and purified from the midguts of viruliferous populations, and used as the template to synthesize the double-strand cDNAs. The cDNAs digested with SfiⅠ were ligated into the library vector pGADT7, and then were transformed into the Escherichia coli DH10B cells to amplify the cDNA library. The cDNA library was calculated the number of transformants per fraction from 1 μL of ligation reaction and the average insert size from the population of 32 released inserts. The results showed the titer of the amplified library was 1.5×106 cfu /mL. The average size of inserts was 1.0~2.0 kb in the cDNA library. The yeast two-hybrid cDNA library derived from the viruliferous WBPH midgut will be useful for the future study on the interaction between insect vector and SRBSDV.
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Received: 04 August 2015
Published: 29 December 2015
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